Project Summary/Abstract The proposed work will provide training to support the candidate's long-term career goal of studying the molecular mechanisms underpinning mitochondrial ultrastructure in cardiac cells. Mitochondrial organization is particularly critical in the heart, as mitochondrial respiration generates the massive amount of energy required for cardiac function. Mitochondrial form and function are intimately linked; cristae, the site of respiration, form aberrant structures in a number of cardiomyopathies and in aged cardiac tissues. The mitochondrial inner membrane (IMM) is laterally organized into distinct functional and morphological domains. However, the molecular mechanisms of IMM organization in cardiac and other cells are largely unknown. In this context, the research goals of this proposal are necessary to understand the basic molecular principles guiding mitochondrial architectural organization. The MICOS complex organizes cristae junctions (CJs), sites along the IMM that delineate the boundary and cristae domains, which house biogenesis and respiratory machinery, respectively. MICOS is critical for regulating the copy number and positioning of CJs and mutations in MICOS lead to a reduction of respiratory function. However, the mechanisms that underlie MICOS assembly and regulation are not well understood. To address these deficits, the candidate will determine the basis of molecular action of the MICOS subunits Mic60 and Mic19 (Specific Aim 1). These proteins are hypothesized, based on the candidate's prior work, to determine CJ copy number and placement. The proposed work will also take candidate and forward approaches to determine mechanisms of regulation of MICOS function (Specific Aims 2 and 3). During the K99 phase of the proposal, the candidate will be trained in protein biochemical analyses and systems biology approaches that will promote the accomplishment of each of these research goals during the independent phase. The candidate has a strong background in organelle biology and the institutional and mentor laboratory training environments of the candidate are world-class and meet the training needs of the candidate, making the short-term and long-term goals of this proposal attainable. Determining the molecular principles guiding IMM organization will enable the candidate to focus on cristae regulation in the heart and identify potential therapeutic approaches for the treatment of cardiac disease, which will be the basis of future grant applications.